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Carbon black (CB) has a wide range of industrial applications and recently has been used as the basic model for environmental health studies on airborne particulate matters (PM). Exposure characterization of CB is always the first and most important step towards a better understanding of its effects on human health. However, efforts were largely limited by the lack of valid methods capable of quantifying CB in biological samples. Here, we developed a new method based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for CB quantification in bio-samples, with a detection limit of 4.3 ng. The method is so economical and convenient that it can be performed in most biology labs. The application of the method was successfully demonstrated in three different cell models (mouse macrophage cells (Raw264.7), human epithelial cells (A549) and mouse mesenchymal stem cells (MSC)) and the results showed that the uptake rates decrease in the order of MSC > Raw264.7 > A549. The surprisingly highest uptake rate of MSC deserves further investigation. The novel method provides a complementary quantitative tool to the use of conventional methods such as radioactive and fluorescent labeling and may facilitate related toxicological studies.

There is a large interest in developing nanoparticles and extracellular vesicles for delivery of therapeutics or imaging agents. Regulatory approval of such products requires knowledge about their biodistribution, metabolism and excretion. We here discuss possibilities and challenges of methods used for such studies, which most often are performed after labelling with radioactive isotopes or fluorescent molecules. It is important to evaluate if the labelled and unlabeled products can be expected to behave similarly in the body. Furthermore, one needs to critically consider whether the labels are still associated with the product at the time of analyses. We discuss advantages and disadvantages of different imaging modalities such as PET, SPECT, MRI, CT, ultrasound and optical imaging for whole-body biodistribution, and describe how to estimate the amount of labelled product in harvested organs and tissue. Microscopy of cells and tissues and various mass spectrometry methods are also discussed in this review. The authors have been supported by the Norwegian Cancer Society, the Research Council of Norway and the South Eastern Norway Regional Health Authority.

A recent publication described the construction and utility of a comprehensive “Chromatic Bacteria” toolbox containing a set of genetic tools that allows for fluorescently tagging a variety of Proteobacteria. In an effort to expand the range of bacteria taggable with the Chromatic Bacteria toolbox, a series of Himar1 transposon vectors was constructed to mediate insertion of fluorescent protein and antibiotic resistant genes. The Himar1 transposon was chosen as it is known to function in a wide range of bacterial species. To test the suitability of the new Himar1 Chromatic Bacteria plasmid derivatives, conjugations were attempted on recently isolated non-model organisms. Although we were unsuccessful in delivering the plasmids into Gram-positive bacterial isolates, we successfully modified previously recalcitrant isolates to the first set of the Chromatic Bacteria toolbox, such as Sphingomonas sp. Leaf357 and Acidovorax sp. Leaf84. This manuscript reports on the currently available plasmids and transposition success in different bacteria.

Target identification (target-ID) is an important step in elucidating the mechanisms of action of bioactive small molecules. In the past few decades, a number of target-ID methods have been developed. Among these, affinity labeling has been reliably used for specific modifications, as well as for the identification of weakly interacting protein targets, membrane-associated protein targets, and target-interacting proteins under native cellular conditions, which are generally difficult to achieve by conventional pull-down methods. In general, affinity labeling utilizes chemical probes composed of a bioactive small molecule, a reactive group, and a detection unit. However, the design and synthesis of highly functionalized chemical probes is often time-consuming. To address this issue, we have recently developed some simple affinity labeling methods using small fluorogenic tags, such as 4-alkoxy-7-nitro-2,1,3-benzoxadiazole (O-NBD), 2,3-dichloromaleimide (diCMI), and 4-azidophthalimide (AzPI), and successfully achieved the specific fluorescent labeling of target proteins, even in living cells. These methods should be useful for target-ID in phenotypic drug discovery.

Generation of three-dimensional (3D) data sets from serial sections of tissues imaged by light microscopy (LM) allows identification of rare structures by morphology or fluorescent labeling. Here, we demonstrate a workflow for correlative LM and electron microscopy (EM) from 3D LM to 3D EM, using the same sectioned material for both methods consecutively. The new approach is easy to reproduce in routine EM laboratories and applicable to a wide range of organs and research questions.

Fluorescent labeling is an established method for visualizing cellular structures and dynamics. The fundamental diffraction limit in image resolution was recently bypassed with the development of super-resolution microscopy. Notably, both localization microscopy and stimulated emission depletion (STED) microscopy impose tight restrictions on the physico-chemical properties of labels. One of them—the requirement for high photostability—can be satisfied by transiently interacting labels: a constant supply of transient labels from a medium replenishes the loss in the signal caused by photobleaching. Moreover, exchangeable tags are less likely to hinder the intrinsic dynamics and cellular functions of labeled molecules. Low-affinity labels may be used both for fixed and living cells in a range of nanoscopy modalities. Nevertheless, the design of optimal labeling and imaging protocols with these novel tags remains tricky. In this review, we highlight the pros and cons of a wide variety of transiently interacting labels. We further discuss the state of the art and future perspectives of low-affinity labeling methods.

The file attached to this record is the author's final peer reviewed version. The Publisher's final version can be found by following the DOI link. Fluorescently labelling proteins such as insulin have wide ranging applications in a pharmaceutical research and drug delivery. Human insulin (Actrapid®) was labelled with fluorescein isothiocyanate (FITC) and the synthesised conjugate identified using reverse phase high performance liquid chromatography (RP-HPLC) on a C18 column and a gradient method with mobile phase A containing 0.1% trifluoroacetic acid (TFA) in Millipore water and mobile phase B containing 90% Acetonitrile, 10% Millipore water and 0.1% TFA. Syntheses were carried out at varying reaction times between 4 and 20 h. Mono-labelled FITC-insulin conjugate was successfully synthesised with labelling at the B1 position on the insulin chain using a molar ratio of 2:1 (FITC:insulin) at a reaction time of 18 h and confirmed by electrospray mass spectroscopy. Reactions were studied across a pH range of 7–9.8 and the quantities switch from mono-labelled to di-labelled FITC-insulin conjugates at a reaction time of 2 h (2:1 molar ratio) at pH > 8. The conjugates isolated from the studies had biological activities in comparison to native insulin of 99.5% monoB1, 78% monoA1, 51% diA1B1 and 0.06% triA1B1B29 in HUVEC cells by examining AKT phosphorylation levels. MonoB1 FITC-insulin conjugate was also compared to native insulin by examining cell surface GLUT4 in C2C12 skeletal muscle cells. No significant difference in the cellular response was observed for monoB1 produced in-house compared to native insulin. Therefore mono-labelled FITC-insulin at the B1 position showed similar biological activity as native insulin and can potentially be used for future biomedical applications.

For decades, we have relied on population and time-averaged snapshots of dynamic molecular scale events to understand how genes are regulated during development and beyond. The advent of techniques to observe single-molecule kinetics in increasingly endogenous contexts, progressing from in vitro studies to living embryos, has revealed how much we have missed. Here, we provide an accessible overview of the rapidly expanding family of technologies for single-molecule tracking (SMT), with the goal of enabling the reader to critically analyse single-molecule studies, as well as to inspire the application of SMT to their own work. We start by overviewing the basics of and motivation for SMT experiments, and the trade-offs involved when optimizing parameters. We then cover key technologies, including fluorescent labelling, excitation and detection optics, localization and tracking algorithms, and data analysis. Finally, we provide a summary of selected recent applications of SMT to study the dynamics of gene regulation.

The relevance of extracellular vesicles (EVs) has grown exponentially, together with innovative basic research branches that feed medical and bioengineering applications. Such attraction has been fostered by the biological roles of EVs, as they carry biomolecules from any cell type to trigger systemic paracrine signaling or to dispose metabolism products. To fulfill their roles, EVs are transported through circulating biofluids, which can be exploited for the administration of therapeutic nanostructures or collected to intercept relevant EV-contained biomarkers. Despite their potential, EVs are ubiquitous and considerably heterogeneous. Therefore, it is fundamental to profile and identify subpopulations of interest. In this study, we optimized EV-labeling protocols on two different high-resolution single-particle platforms, the NanoFCM NanoAnalyzer (nFCM) and Particle Metrix ZetaView Fluorescence Nanoparticle Tracking Analyzer (F-NTA). In addition to the information obtained by particles’ scattered light, purified and non-purified EVs from different cell sources were fluorescently stained with combinations of specific dyes and antibodies to facilitate their identification and characterization. Despite the validity and compatibility of EV-labeling strategies, they should be optimized for each platform. Since EVs can be easily confounded with similar-sized nanoparticles, it is imperative to control instrument settings and the specificity of staining protocols in order to conduct a rigorous and informative analysis.

Although largely overlooked compared to bacterial infections, fungal infections pose a significant threat to the health of humans and other organisms. Many pathogenic fungi, especially Candida species, are extremely versatile and flexible in adapting to various host niches and stressful situations. This leads to high pathogenicity and increasing resistance to existing drugs. Due to the high level of conservation between fungi and mammalian cells, it is hard to find fungus-specific drug targets for novel therapy development. In this respect, it is vital to understand how these fungi function on a molecular, cellular as well as organismal level. Fluorescence imaging allows for detailed analysis of molecular mechanisms, cellular structures and interactions on different levels. In this manuscript, we provide researchers with an elaborate and contemporary overview of fluorescence techniques that can be used to study fungal pathogens. We focus on the available fluorescent labelling techniques and guide our readers through the different relevant applications of fluorescent imaging, from subcellular events to multispecies interactions and diagnostics. As well as cautioning researchers for potential challenges and obstacles, we offer hands-on tips and tricks for efficient experimentation and share our expert-view on future developments and possible improvements., Fungal infections such as those caused by Candida species impose serious health problems to patients, and using state-of-the-art fluorescence techniques, it is possible to pave the way towards novel, highly-needed, druggable targets.

Nature harnesses the unique properties of cysteinyl radical intermediates for a diverse range of essential biological transformations including DNA biosynthesis and repair, metabolism, and biological photochemistry. In parallel, the synthetic accessibility and redox chemistry of cysteinyl radicals renders them versatile reactive intermediates for use in a vast array of synthetic applications such as lipidation, glycosylation and fluorescent labelling of proteins, peptide macrocyclization and stapling, desulfurisation of peptides and proteins, and development of novel therapeutics. This review provides the reader with an overview of the role of cysteinyl radical intermediates in both chemical synthesis and biological systems, with a critical focus on mechanistic details. Direct insights from biological systems, where applied to chemical synthesis, are highlighted and potential avenues from nature which are yet to be explored synthetically are presented.

The efficiency of injectable biomaterials as minimally invasive therapeutics significantly relies on biomaterial's characteristics, such as stability, biodegradation rate, and interaction with the host tissue, which requires real‐time tracking of the biomaterials. Fluorescence imaging is considered as a noninvasive technique for monitoring biomaterials; however, the commonly used fluorescent agents are often accompanied by photobleaching and toxicity. Herein, graphene quantum dots (GQDs) are introduced as a biocompatible and stable fluorophore for imaging and noninvasive monitoring of a physically cross‐linked injectable shear‐thinning biomaterial (STB) of gelatin–silicate nanoplatelets. Silicate nanoplatelets and GQDs serve as the physical cross‐linkers of gelatin making electrostatic interaction with gelatin chains. Different STB‐GQDs formulations are assessed in terms of fluorescence intensity, injectability, thermal stability, and cellular biocompatibility. STB‐GQDs with 0.06% GQDs, 6% solid material, and 50% silicate in the solid material show the strongest in vitro fluorescence and the highest thermal stability. In vivo monitoring of STB‐GQDs is also achieved through fluorescent imaging where incorporated GQDs exhibit a robust and stable signal, suggesting their promising applications in long‐term tracking of gelatin‐based STBs.

Single-molecule localization microscopy (SMLM) describes a family of powerful imaging techniques that dramatically improve spatial resolution over standard, diffraction-limited microscopy techniques and can image biological structures at the molecular scale. In SMLM, individual fluorescent molecules are computationally localized from diffraction-limited image sequences and the localizations are used to generate a super-resolution image or a time course of super-resolution images, or to define molecular trajectories. In this Primer, we introduce the basic principles of SMLM techniques before describing the main experimental considerations when performing SMLM, including fluorescent labelling, sample preparation, hardware requirements and image acquisition in fixed and live cells. We then explain how low-resolution image sequences are computationally processed to reconstruct super-resolution images and/or extract quantitative information, and highlight a selection of biological discoveries enabled by SMLM and closely related methods. We discuss some of the main limitations and potential artefacts of SMLM, as well as ways to alleviate them. Finally, we present an outlook on advanced techniques and promising new developments in the fast-evolving field of SMLM. We hope that this Primer will be a useful reference for both newcomers and practitioners of SMLM. This Primer explains the central concepts of single-molecule localization microscopy (SMLM) before discussing experimental considerations regarding fluorophores, optics and data acquisition, processing and analysis. The Primer further describes recent high-impact discoveries made by SMLM techniques and concludes by discussing emerging methodologies.

Photochemical properties of a new bis-cyanine dye have been investigated, including spectral kinetic characteristics of singlet and triplet excited states as well as affinity to BSA as a representative blood protein. The results obtained reveal, that the dye is a promising agent for imaging and fluorescent labeling of biomacromolecules.

Flow cytometric analysis and sorting rely on fluorescent labeling with multiple antibody-fluorochrome conjugates. Currently available conjugates remain on the cells after flow sorting and thus can limit downstream applications. In contrast, REAlease® conjugates enable specific cell labeling and can be completely removed from the epitopes after the experiment; cell epitopes and fluorescence channels that were blocked through labeling become available for subsequent experiments.

This article describes a newly devised autosomal short tandem repeat (STR) multiplex polymerase chain reaction (PCR) system for 19 autosomal loci (D12S391, D13S317, D16S539, D18S51, D19S433, D2S1338, D21S11, D3S1358, D5S818, D6S1043, D7S820, D8S1179, CSF1PO, FGA, TH01, TPOX, vWA, Penta D and Penta E), 27 Y-chromosome STR loci (DYS19, DYS385, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS448, DYS449, DYS456, DYS458, DYS460, DYS481, DYS518, DYS533, DYS570, DYS576, DYS635, DYS627, YGATAH4 and DYF387S1) and amelogenin with six-colour fluorescent labelling. Various parameters were evaluated, such as its accuracy, sensitivity, specificity, stability, ability to analysis of mixtures and effects of changes in the PCR-based procedures. All of the 47 selected STR loci were accurately and robustly amplified from 282 bloodstain samples. The species-specificity was high and some ability to inhibit Hematin was identified. The lowest detectable DNA amount was ≥0.125 ng. All of the male loci of the secondary component were revealed precisely when the control DNA was mixed at male/female and male/male ratios of 1:4 or more. We conclude that the present 19-plex autosomal STR and 27 Y-STR assay is both accurate and sensitive. It constitutes an additional powerful tool for forensic applications.

We investigated the thermoresponsive behavior of poly(acrylamide-co-acrylonitrile) (poly(AAm-co-AN)) with an upper critical solution temperature (UCST) in water to apply this polymer in cosmetic pr...

Traditionally, scientists studied microbiology through the manner of batch cultures, to conclude the dynamics or outputs by averaging all individuals. However, as the researches go further, the heterogeneities among the individuals have been proven to be crucial for the population dynamics and fates. Due to the limit of technology, single-cell analysis methods were not widely used to decipher the inherent connections between individual cells and populations. Since the early decades of this century, the rapid development of microfluidics, fluorescent labelling, next-generation sequencing, and high-resolution microscopy have speeded up the development of single-cell technologies and further facilitated the applications of these technologies on bacterial analysis. In this review, we summarized the recent processes of single-cell technologies applied in bacterial analysis in terms of intracellular characteristics, cell physiology dynamics, and group behaviors, and discussed how single-cell technologies could be more applicable for future bacterial researches.

Metarhizium anisopliae, an entomopathogenic fungus, is found to contribute to plant nutrient uptake and condidered as a plant endophyte. However, there is very little visual evidence that d...

In recent years, due to the development of the field of fluorescent labeling and the importance of hydrogen sulfide (H2S) in biological systems, H2S fluorescence detection and bio-imaging have become popular. Especially, near-infrared or ratiometric fluorescence probe are booming because of their respective superiority. In this work, we integrated NIR and ratiometric (17-fold I650/I565) fluorescent emissions into probe molecular to detect H2S in pH 7.4 PBS buffer solution based on ICT lighten with high sensitivity (detection limit 1.08 nM) and fast response time (less than 120 s). In addition, the detection process can be monitored by naked-eye (from yellow to red). Thus the probe can image the concentration distribution of H2S in living cancer cells. NIR fluorescent probe has good tissue penetration, which was successfully applied in mice imaging.

Herein, we present a synthesis of a molecular construct based on the polysulfonated indocarbocyanine dye and the lysine. The chemical structure of the construct obtained enables fluorescent labeling of oligonucleotide probes designed for the immobilization on the oligonucleotide biochips.

Receptor for activated C kinase 1 (RACK1) is a eukaryote-specific ribosomal protein implicated in diverse biological functions. To engineer ribosomes for specific fluorescent labeling, we selected RACK1 as an target given its location on the small ribosomal subunit and other properties. However, prior results suggested that RACK1 has roles both on and off the ribosome, and such an exchange might be related to its various cellular functions and hinder our ability to use RACK1 as a stable fluorescent tag for the ribosome. In addition, the kinetics of spontaneous exchange of RACK1 or any ribosomal protein from a mature ribosomein vitroremain unclear. To address these issues, we engineered fluorescently-labeled human ribosomes via RACK1, and applied bulk and single-molecule biochemical analyses to track RACK1 on and off the human ribosome. Our results demonstrate that, despite its cellular non-essentiality from yeast to humans, RACK1 readily re-associates with the ribosome, displays limited conformational dynamics, and remains stably bound to the ribosome for hoursin vitro. This work sheds insight onto the biochemical basis of ribosomal protein exchange on and off a mature ribosome and provides tools for single-molecule analysis of human translation.

Sialylation can influence the stability, half-life, and immunogenicity of glycoproteins, but sialylated N-glycans are known to be difficult to analyze. Human alpha1-acid glycoprotein (AGP) is reported to have glycans that consist of sialylated N-glycans. The N-glycan profiling of AGP is qualitatively and quantitatively investigated here by UPLC and LC-ESI-MS/MS. Three fluorescent tags (AB, AA, and ProA) and two separation columns (HILIC and AEX-HILIC) were adopted to confirm and compare each analytical characteristic. The results of AA were comparable to those of the well-established AB. The qualification of ProA was notable due to its superior fluorescence intensity and ionization efficiency, and ProA showed smaller quantitative or larger-sized fragments in LC-ESI-MS/MS compared to AB and AA. However, the MS quantification of ProA was distorted because the increased sialylation level decreased the LC-ESI-MS/MS ionization efficiency. HILIC had better peak separability, AEX-HILIC had an advantage in UPLC sialylation profiling, and each isomeric glycan could be identified by both columns in LC-ESI-MS/MS. In conclusion, ProA is favored for UPLC and LC-ESI-MS/MS detection but not reliable for MS quantification. This study firstly demonstrates the qualification and quantification of sialylated N-glycans by comparing the commonly used analytical conditions with different fluorescent tags, columns, and instruments.

Fluorescence imaging has facilitated fluorescent probes to analyze the subcellular localization and dynamics of biological targets. In this paper, we reported a fluorogenic probe for bacteria imaging. The probe was an imidazolium-derived pyrene compound, which self-assembled to form nano-particles and the pyrene fluorescence was quenched by the aggregation effects. When the self-assembly nano-particles interacted with anionic bacteria surfaces, synergistic effects of electrostatic interaction and hydrophobic force caused competing binding between bacteria surfaces and imidazoliums. This binding resulted in the disassembly of the aggregates to give fluorescence turn-on signal. Meanwhile, the probe bound bacteria surfaces and displayed both pyrene-excimer and pyrene-monomer fluorescence, which gave ratiometric signal. Then, fluorescent labeling by the probe enabled the two-photo ratiometric imaging of bacteria.

Track-etched PET membranes (TMs) are one of the first commercially available nanostructured materials. The number, size and shape of pores (or nanochannels) can be carefully tailored at industrial scale. Selective pore modification can be achieved using remnant radicals after the etching process; however, this technique has some limitations. In this work, a simple and straightforward procedure is described for selectively grafting glycidyl methacrylate (GMA) and acrylic acid into the nanochannel walls of track-etched PET membranes. Grafting polymerization was carried out at short and long post-etching time by thermal initiation. The ultra-low amounts of grafting modification were measured by fluorescence labelling and membrane hydrolysis. The presence/absence of polyGMA and polyacrylic acid was confirmed by FTIR spectroscopy and MS-ESI. Peroxide compounds were detected in track-etched membranes and attributed responsibility for the selective grafting polymerization in the nanochannel walls of TMs. This simple and quick procedure will enhance the development of novel functional materials based on this technology. Fil: Aguiar, Constanza. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Grupo Vinculado al IMBICE - Grupo de Biología Estructural y Biotecnología-Universidad Nacional de Quilmes - GBEyB | Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Grupo Vinculado al IMBICE - Grupo de Biología Estructural y Biotecnología-Universidad Nacional de Quilmes - GBEyB | Universidad Nacional de la Plata. Instituto Multidisciplinario de Biología Celular. Grupo Vinculado al IMBICE - Grupo de Biología Estructural y Biotecnología-Universidad Nacional de Quilmes - GBEyB; Argentina Fil: Soto Espinoza, Silvia Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Grupo Vinculado al IMBICE - Grupo de Biología Estructural y Biotecnología-Universidad Nacional de Quilmes - GBEyB | Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Grupo Vinculado al IMBICE - Grupo de Biología Estructural y Biotecnología-Universidad Nacional de Quilmes - GBEyB | Universidad Nacional de la Plata. Instituto Multidisciplinario de Biología Celular. Grupo Vinculado al IMBICE - Grupo de Biología Estructural y Biotecnología-Universidad Nacional de Quilmes - GBEyB; Argentina Fil: Laurella, Sergio Luis. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Departamento de Química. Laboratorio de Estudio de Compuestos Orgánicos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Grasselli, Mariano. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Grupo Vinculado al IMBICE - Grupo de Biología Estructural y Biotecnología-Universidad Nacional de Quilmes - GBEyB | Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Grupo Vinculado al IMBICE - Grupo de Biología Estructural y Biotecnología-Universidad Nacional de Quilmes - GBEyB | Universidad Nacional de la Plata. Instituto Multidisciplinario de Biología Celular. Grupo Vinculado al IMBICE - Grupo de Biología Estructural y Biotecnología-Universidad Nacional de Quilmes - GBEyB; Argentina

Among biocompatible materials, block copolymers (BCPs) possess several advantages due to the control of their chemistry and the possibility of combining various blocks with defined properties. Consequently, BCPs drew considerable attention as biocompatible materials in the fields of drug delivery, medicine and bioimaging. Fluorescent labeling of BCPs quickly appeared to be a method of choice to image and track these materials in order to better understand the nature of their interactions with biological media. However, incorporating fluorescent markers (FM) into BCPs can appear tricky; we thus intend to help chemists in this endeavor by reviewing recent advances made in the last 10 years. With the choice of the FM being of prior importance, we first reviewed their photophysical properties and functionalities for optimal labeling and imaging. In the second part the different chemical approaches that have been used in the literature to fluorescently label BCPs have been reviewed. We also report and discuss relevant applications of fluorescent BCPs in bioimaging., This review describes the different chemical approaches and strategies to fluorescently label block copolymers. The review also focuses on the properties of fluorescent markers and the bioimaging applications allowed by the labeling.

Three environment-sensitive probes were developed for the hERG channel based on the nitrobenzoxadiazole fluorophore herein. After careful evaluation, probes M1 and M3 were found to have a high affinity for imaging the hERG channel in the cell-based experiment. Compared with other fluorescent labeling technologies (such as fluorescent proteins), these probes afford a convenient and economical method to determine hERG channel in vitro and in cellulo. Therefore, these probes are expected to be applicable for usage in physiological and pathological studies of hERG channels and have the potential to establish a screening system for hERG channels.

Two novel, water-soluble, merocyanine fluorophores were readily prepared by microwave-assisted synthesis. Full optical characterization was performed in a series of protic and aprotic solvents, and the dyes displayed fluorescence in the red region with up to a 20-fold decrease in brightness in water, demonstrating a strong environmental sensitivity hereby termed as solvato-fluorogenicity (to distinguish from solvatochromism). Shorter fluorescent lifetimes were also measured in water, which confirmed this character. These dyes were conjugated to a modified polymyxin scaffold that allowed fluorescence “switch-on” upon binding to Gram-negative bacterial membranes, and selective fluorescence detection of bacteria in a wash-free protocol.

Nanotechnologies hold great promise for various applications. To predict and guarantee the safety of novel nanomaterials, it is essential to understand their mechanism of action in an organism, causally connecting adverse outcomes with early molecular events. They are best investigated using non-invasive advanced optical methods, such as high-resolution live-cell fluorescence microscopy, which require stable labelling of nanoparticles with fluorescent dyes. When performed inadequately, unbound fluorophores and inadvertently altered chemical and physical properties of the nanoparticles can, however, result in experimental artefacts and erroneous conclusions.To prevent such unintentional errors, we here describe a minimal combination of experimental methods to enable artefact-free fluorescent labelling of metal-oxide nanoparticles – the largest subpopulation of nanoparticles by industrial production and applications – and demonstrate its application in the case of TiO2 nanotubes. We 1) characterize potential changes of the nanoparticles’ surface charge and morphology that might occur during labelling, and 2) assess stable binding of the fluorescent dye to nanomaterial, which ensures correct nanoparticle localization. Together, these steps warrant the reliability and reproducibility of advanced optical tracking, which is necessary to explore nanomaterials’ mechanism of action and will foster widespread and safe use of new nanomaterials.Abstract Figure

In this contribution, fluorescence spectroscopy based on G-quadruplex formation was developed for the quantification of ATP in cell. In the absence of ATP, thioflavin T (ThT) dye can combine with the G-rich ATP aptamer to form an ATP aptamer-ThT G-quandruplex complex, resulting in the enhancement of fluorescence intensity; otherwise, fluorescence intensity of the system will weaken with the addition of ATP, because ATP has a strong affinity with G-rich ATP aptamer and can replace ThT to form an ATP aptamer-ATP complex. A calibration model based on generalized ratio quantitative analysis model was employed to mitigate the influence of scatterers and background absorbers in cell suspensions. The proposed fluorescence method was applied to the quantitative analysis of ATP in two type of cell lines, and achieved satisfactory quantitative results with accuracy comparable to that of the reference method–ATP detection kit. The limit of detection and limit of quantification of the proposed method for ATP in cell were estimated to be 0.22 and 0.66 μM, respectively. This proposed fluorescence method is highly simple and rapid, and does not require the use of fluorescent labeling.

A biocompatible, nontoxic theranostic nanoplatform consisting of mesoporous silica-coated ferroferric oxide (Fe3O4) and Mn-doped ZnS-ZnS quantum dots has been synthesized via a layer-by-layer method. Transmission electron microscopy, X-ray diffractometer, magnetometry, and fluorophotometer were employed to characterize the nanoplatform. The nanoplatform exhibited excellent superparamagnetic, fluorescent and light adsorption properties. A template method was introduced to form a mesoporous silica structure on the nanoplatform, lowering the mass of the nanoplatform and effectively promoting the absorption of incident light, compared with traditional silica layer. In addition, after endocytosis of the nanoplatform, cancer cells were easily detected under fluorescence microscope because of the excellent fluorescent behavior of quantum dos. Moreover, vitro cell experiments confirmed that nanoplatform possessed perfect photothermal effect to destroy tumor cells under the laser irradition. Therefore, ferroferric oxide/quantum dots nanoplatforms, combined with fluorescent labeling and photothermal therapy for cancer cells, are expected to be a promising biopotential materials in the field of diagnosis and treatment.

12 pags., 2 figs., 1 tab., 4 schs. -- This article belongs to the Special Issue Exclusive Feature Papers in Colorants, A convergent synthetic route to a tetrasaccharide related to PI-88, which allows the incorporation of a fluorescent BODIPY-label at the reducing-end, has been developed. The strategy, which features the use of 1,2-methyl orthoesters (MeOEs) as glycosyl donors, illustrates the usefulness of suitably-designed BODIPY dyes as glycosyl labels in synthetic strategies towards fluorescently-tagged oligosaccharides, This research was funded by Spanish MINISTERIO DE ECONOMIA Y COMPETITIVIDAD, GOBIERNO DE ESPAÑA (projects MAT2017–83856-C3-1-P and 3-P, PiD2020-1147555GB-C33), the MINISTERIO DE CIENCIA INNOVACION Y UNIVERSIDADES (project RTI2018-094862-B-I00), and the GOBIERNO VASCO (project IT912-16)

In order to grasp the germplasm resources of walnut in Yunnan province, the genetic diversity and relationship of 21 walnuts varieties commonly planted in Yunnan province were analyzed using SSR fluorescent labeling technology. The results showed that the average number of allelic loci of 12 pairs of primers used in 21 walnut species samples was 7.916 7. The mean number of alleles, Shannon Wiener index (I) and polymorphic information content (PIC) per locus were 4.3516, 1.643 6 and 0.705 5. Twelve pairs of polymorphism information content (PIC) were higher than 0.25, indicating that the 12 pairs of primers had good polymorphism in the samples. In the phylogenetic analysis, the 21 walnut varieties were divided into 7 subgroups, indicating that the 21 walnut varieties had rich genetic diversity, extensive gene exchange and complex genetic background in the breeding process.

Biomolecular condensates are membrane-less cellular compartments that form via phase separation. They serve a multitude of functions in all types of cells. Important insights into the composition, architecture and dynamics of biomolecular condensates have been obtained by harnessing the power of fluorescence-based technologies. In this chapter, methods will be discussed for (1) fluorescent labelling of macromolecules, (2) spatial and temporal mapping and tracking of target molecules in cellular and in vitro settings, (3) controlling formation and dissolution of biomolecular condensates, and (4) fluorescence-based condensate-targeted drug discovery.

Despite the urgent needs of imaging living specimens for cutting-edge biological research, most of the existing fluorescent labeling methods suffer from either poor optical properties or complicated operations to realize cell-permeability and specificity. Here, we introduce a method to overcome this tradeoff by incubating living cells and tissues with bright and photostable fluorescent dyes, no matter if they are cell-permeable or not, at particular conditions (concentration and temperature) without physical cell-penetration or chemical modifications. Particularly, using Atto 647N to replace the most-commonly-used red-absorbing SiR dye in live-cell long-term imaging, we obtain 2.5-time enhancement on fluorescence brightness and photostability. These improvements give access to the discovery of a new interaction model between endoplasmic reticulum and mitochondria. These results indicate the great potential of using dyes, which are normally considered “live-cell incompatible”, to capture the morphology and dynamics of subcellular structures in living specimens. Our strategy has expanded the scientist’s toolbox for understanding the dynamics and interactions of subcellular structures in living specimens.

Neuronal loss is at the core of many neuropathologies, including stroke, Alzheimer's disease, and Parkinson's disease. Different methods were developed to study the process of neuronal survival upon cytotoxic stress. Most methods are based on biochemical approaches that do not allow single-cell resolution or involve complex and costly methodologies. Presented here is a versatile, inexpensive, and effective experimental paradigm to study neuronal survival. This method takes advantage of sparse fluorescent labeling of the neurons followed by live imaging and automated quantification. To this aim, the neurons are electroporated to express fluorescent markers and co-cultured with non-electroporated neurons to easily regulate cell density and increase survival. Sparse labeling by electroporation allows a simple and robust automated quantification. In addition, fluorescent labeling can be combined with the co-expression of a gene of interest to study specific molecular pathways. Here, we present a model of stroke as a neurotoxic model, namely, the oxygen-glucose deprivation (OGD) assay, which was performed in an affordable and robust homemade hypoxic chamber. Finally, two different workflows are described using IN Cell Analyzer 2200 or the open-source ImageJ for image analysis for semi-automatic data processing. This workflow can be easily adapted to different experimental models of toxicity and scaled up for high-throughput screening. In conclusion, the described protocol provides an approachable, affordable, and effective in vitro model of neurotoxicity, which can be suitable for testing the roles of specific genes and pathways in live imaging and for high-throughput drug screening.

The nuclear envelope (NE), a double membrane that separates nuclear components from the cytoplasm, undergoes a breakdown and reformation during cell division. To trace NE dynamics, the NE needs to be labeled with a fluorescent marker, and for this purpose, markers based on inner nuclear membrane (INM) proteins are normally used. However, NE labeling with INM proteins has some limitations. Here, we introduce a protocol for fluorescent labeling and imaging of NE that does not rely on INM proteins, along with protocols for simultaneously imaging two nuclear components and for time-lapse imaging of labeled cells.

Observing the localization, the concentration, and the distribution of proteins in cells or organisms is essential to understand theirs functions. General and versatile methods allowing multiplexed imaging of proteins under a large variety of experimental conditions are thus essential for deciphering the inner workings of cells and organisms. Here, we present a general method based on the non-covalent labeling of a small protein tag, named FAST (fluorescence-activating and absorption-shifting tag), with various fluorogenic ligands that light up upon labeling, which makes the simple, robust, and versatile on-demand labeling of fusion proteins in a wide range of experimental systems possible.